Trends and antibiotic susceptibility patterns of diarrhoeal pathogens – experience over 14 years in southern India

Abstract Introduction. Enteric pathogens contribute significantly to morbidity in a developing country such as India. Early and prompt diagnosis of diarrhoeal diseases can reduce the mortality rate, particularly in children. The pattern of sensitivity to antimicrobials for the common pathogens can vary from time to time. The present study was conducted to study the pathogen distribution and antimicrobial susceptibility pattern during the study period (January 2010 to December 2023). Hypothesis/gap statement. Studying the changing trend in the antimicrobial sensitivity pattern of diarrhoeal pathogens over a decade can help to plan future treatment options. Aim. This study was undertaken to provide insights into the changing pattern of pathogen distribution and antimicrobial susceptibility for enteric pathogens over 14 years. Methods. A retrospective observational cohort analysis was conducted on all the stool pathogens isolated from the samples received in the microbiology department of a tertiary care hospital from 2010 to 2023. The demographic details, stool microscopy, culture reports, and antimicrobial susceptibility patterns were noted. Results. A total of 18 336 stool specimens were received in the microbiology laboratory between January 2010 and December 2023, of which 1354 specimens had diarrhoeal pathogens grown in culture. Out of these 1354 specimens, 591 (44%) had Salmonella, 471 (35%) Shigella, 181 (13%) Vibrio cholerae, and 80 (6%) Aeromonas species. Among these pathogens, susceptibility to ceftriaxone was seen in 93% (552 isolates) of Salmonella species, 89% (420 isolates) of Shigella species, and 95% (171 isolates) of Vibrio cholerae; 91% (73 isolates) of Aeromonas species were susceptible to chloramphenicol. Some major parasites were also observed on microscopy. Conclusion. Timely diagnosis of diarrhoeal pathogens can be life-saving for patients at the extremes of age, i.e. in children and the elderly. Pathogens can exhibit a changing susceptibility pattern to antibiotics, which should be regularly observed to plan future therapy.


INTRODUCTION
Gastroenteritis is considered to be an important health problem in India.It is a condition caused by bacteria, viruses, and parasites [1] and is a major cause of illness and death in various age groups, especially at the extremes of age groups, i.e. in children and the elderly [2].This can be attributed to poor sanitary conditions, improper personal hygiene, and lack of proper homes.Natural calamities such as floods, cyclones, and rainstorms can worsen the situation.Nearly 90% of clinical cases of gastroenteritis are due to polluted drinking water, improper sanitation, and poor hygiene [3], associated with an ever-increasing multidrug resistance among diarrhoeal pathogens due to the indiscriminate use of antimicrobials.Ironically, amidst the rise and outbreak of resistant strains, the emergence of strains susceptible to older antibiotics has been noted [4].Most diarrhoea cases can be managed with hydration and antibiotics are only indicated in certain situations and should be selected appropriately for the target pathogen [5].It is important to study the distribution of enteric pathogens and their antibiotic susceptibility pattern over time in a geographical area to collect information about the common pathogens in that area.This can help formulate the antibiotic policy for empirical therapy and prevent complications by providing early treatment.Traditionally, fluoroquinolones have been used for treating diarrhoea, but due to the development of resistance, azithromycin and doxycycline have replaced them.Third-generation cephalosporins are currently considered the drug of choice for Shigella and Salmonella species, while doxycycline is the drug of choice for for Vibrio cholerae.The current study aims to study the organism profiles and antibiograms of the isolates from stool samples from cases of diarrhoea/dysentery in a tertiary care hospital.This study highlights the emerging trend in the pathogen distribution and antibiotic susceptibility of diarrhoeal isolates as well as stool parasites from 2010 to 2023.

Study design
A retrospective observational cohort analysis was conducted on all the stool pathogens isolated from the samples received from January 2010 to December 2023 with approval from the Institutional Ethics Committee (JIP/IEC/2017/0130).

Study setting
This study was conducted in a tertiary care hospital, located in the southern part of India.

Data collection
The data were collected retrospectively from the laboratory records and the laboratory information system.The demographic details, clinical history of the patients having significant microscopic findings, and pathogens in the stool samples were recorded.A standard protocol for stool processing for culture was used, employing a selective medium (xylose lysine deoxycholate agar), less selective cum differential medium (MacConkey agar), and an enrichment broth (selenite F broth); suspected colonies were screened using catalase test, oxidase test, indole detection, citrate, urea, Kligler iron agar, lysine iron agar, and mannitol motility medium.In the case of suspected cholera cases, thiosulphate citrate bile salt sucrose agar (TCBS) was also used and the specimen was inoculated into alkaline peptone water (APW).Antibiotic sensitivity testing was performed using the Kirby-Bauer technique for a panel of antibiotics (ampicillin, ceftriaxone, cotrimoxazole, cefixime, chloramphenicol, tetracycline, ciprofloxacin) and interpreted as per the CLSI M100 guidelines for Salmonella and Shigella and the CLSI M45 guidelines for Vibrio and Aeromonas for the respective years.The bacterial profile and antimicrobial susceptibility pattern of the bacteria, namely Salmonella species, Vibrio species, Shigella species, and Aeromonas species, was noted.Plesiomonas shigelloides, Edwardsiella tarda, Comamonas aquatica, and Yersinia enterocolitica were also included.Identification of Salmonella species, Vibrio species, Aeromonas species, P. shigelloides, E. tarda, and C. aquatica was simplified after the introduction of MALDI-TOF MS (bioMérieux, France) in 2019.
Serogrouping of Shigella, Salmonella and V. cholerae was performed using corresponding antisera (BD Diagnostics).Parasites detected were also noted.

Statistical analysis
The extended Mantel-Haenszel chi-square test was applied to observe the significance in the various age groups considering the absolute count of the organisms present and absent in a particular age group.
Antibiotic sensitivity patterns for the duration of the study period (14 years) are shown as linear trends in the form of line graphs.

RESULTS
A total of 18 336 stool specimens were received in the microbiology laboratory between January 2010 to December 2023.A total of 591 Salmonella, 471 Shigella, 181 V. cholerae and 80 Aeromonas organisms were isolated.The pathogen-wise distribution is shown in Table 1 and Fig. 1.
The age-wise distribution of pathogens is shown in Table 2 and Fig. 2. The trend over the age groups was significant for V. cholerae (P=0.0000001 by chi-square test) and Shigella species (P=0.0000001 by chi-square test) while it was insignificant for Salmonella and Aeromonas species.
Most of the Salmonella isolates were susceptible to ceftriaxone (93%), with a decreased susceptibility to nalidixic acid and furazolidone (Table 3 and Fig. 3).This was observed in the initial years (2010-2012), following which these antibiotics were abandoned because of increasing resistance.
Most of the Shigella species were susceptible to ceftriaxone (92%), with a few being resistant to it (Table 4 and Fig. 4).Among the V. cholerae isolates, the majority were susceptible to ceftriaxone (94%) (Table 5 and Fig. 5), while 91% (73/80) of the Aeromonas species were susceptible to chloramphenicol (Table 6 and Fig. 6).

DISCUSSION
The present study is a retrospective analysis of the data obtained from 18 336 stool specimens received in the Department of Microbiology from 2010 to 2023.The demographic details, clinical history of the patients having significant microscopic findings, and pathogens in the stool samples were recorded.The bacterial profile and antimicrobial susceptibility pattern of the bacteria were noted.A total of 591 Salmonella, 471 Shigella, 181 Vibrio, and 80 Aeromonas species were isolated.
Salmonella, Shigella, and Vibrio were common in children <10 years of age.In a study by Anjeeta et al., the highest occurrence of diarrhoeal disease was reported among children aged 9-12 years (71.4%), while the lowest incidence was seen in the age group 5-8 years (33.3%)[6].An increase in the incidence of diarrhoeal diseases with an increase in the age of the children was noted.This could be due to improper hand washing habits of the children as well as improper sanitary habits of caretakers after changing the napkins of the children.For most of these isolates, decreased susceptibility to nalidixic acid and furazolidone led to the discontinuation of these drugs for treatment.The trend with the different organisms is discussed subsequently.

Trends in the Salmonella species
Nontyphoidal Salmonella (NTS) infections are a common cause of diarrhoeal disease and are responsible for causing 93 million enteric infections and 155 000 diarrhoeal deaths yearly [7].Most of the NTS are usually associated with selflimiting gastroenteritis, which does not require antimicrobial treatment, but there has been evidence of invasive diseases such as bloodstream infections being caused by them in patients with underlying comorbidities [8].This necessitates the institution of antimicrobial therapy in cases of immunocompromised individuals with co-morbidities.Chloramphenicol, ampicillin, and trimethoprim/sulphamethoxazole (cotrimoxazole) were the drugs of choice for the treatment of salmonellosis in the past [9].In our study, resistance to conventional classes of antibiotics such as chloramphenicol, nalidixic acid,  and furazolidone was observed.Interestingly, NTS that were initially resistant to cephalosporins and fluoroquinolones have shown a diminishing trend of resistance to these antibiotics in recent years [10], and a similar finding was also observed in our isolates.In a study performed by Jacob et al. in southern India, resistance to nalidixic acid (27%) was most common among the tested NTS, followed by resistance to ampicillin (19.3%), cotrimoxazole (14%), ciprofloxacin (12.4%), and chloramphenicol (4%), which is similar to the findings of our study.Ceftriaxone had the highest sensitivity [11].The resistance to beta-lactam antibiotics such as ceftriaxone in this organism is remarkable.This is caused by extended-spectrum beta-lactamases (ESBLs), predominantly CTX-M-15 and SHV-12, being present in those strains [12,13].An ACC-1 AmpC Salmonella Typhi was identified in the blood culture of a 14-year-old girl from our setting in the past.It could have been acquired from drug-resistant bowel flora and atypical resistance was seen with it [14].Genotypic diversity has been observed among the strains present in our region by pulsed-field gel electrophoresis, indicating the existence of many different clones of S. Typhi universally [15].

Trends in the Shigella species
Shigellosis can be life-threatening in populations at the extremes of age (young, elderly).In most communities, the incidence of shigellosis is higher in the summer months due to reduced hand hygiene habits, but a significant peak is also noted during the rainy season [16].In our geographical region, Shigella flexneri was found to be the most common followed by Shigella sonnei, Shigella boydii, and Shigella dysenteriae [17].Cefixime became the drug of choice for shigellosis in our centre from 2012 onwards due to the development of resistance to ciprofloxacin, co-trimoxazole, and ampicillin.Such a resistance pattern points to the ability of Shigella to survive and replicate in the human gut and incorporate externally derived genetic material, including antimicrobial resistance (AMR) genes on transposons from other Gram-negative bacteria [18].With the ease of its availability as well as administration, ciprofloxacin was used by the majority of clinicians, which has contributed to resistance.The pattern of resistance in our study was nalidixic acid followed by cotrimoxazole, ciprofloxacin, ampicillin, and then ceftriaxone, in order of decreasing resistance.We observed a high resistance to ciprofloxacin, as reported in an earlier study from India [19].
Virulence genes have shown diversity in all serotypes of S. flexneri.IpaH (invasion plasmid antigen H) and ial (invasion-associated locus) genes were also detected in almost all of the isolates [17].

Trends in the V. cholerae
Cholera epidemics caused by toxigenic V. cholerae pose a major threat in most developing countries [27].Globally, epidemics of cholera have been reported in 47 countries [28].Outbreaks were observed during the rainy season.
In Kolkata, India, during two consecutive cholera seasons (1989-1990), the Inaba serotype was prevalent, but in our study period  the Ogawa serotype was predominant [29].Similar genetic conversions for cholera have been reported from other parts of the world.A comparison of contemporary reports of antibiotic sensitivity patterns for V. cholerae strains with those isolated in the past shows a vast difference in the antimicrobial resistance profile.The reversal of the antimicrobial susceptibility pattern is due to the decreased use of conventional drugs that had encountered resistance earlier [30].In the current study, reduced susceptibility to the earlier drugs, such as co-trimoxazole, was observed.In a study performed in the same setup from 2008 to 2013, the prevalence of SXT element conserved genes (int, eex, att-prfC, and setR) was noted in all of the isolates.The spread of Haitian-like traits with a creeping MIC (0.75-2 µg m −1 ) for azithromycin was also noted [31].A study performed in different parts of Odisha from 2004 to 2013 also showed decreased susceptibility to co-trimoxazole [32].Sensitivity to   ciprofloxacin was the highest, making it a drug of choice, and the sensitivity to tetracyclines showed a cyclical trend in our study, similar to a study performed in Bangladesh [33].
A meta-analysis performed by Yuan et al. showed a low resistance rate against some antibiotics, including fluoroquinolones, gentamicin, ceftriaxone, doxycycline, kanamycin, and cefotaxime, similar to our study [34].Antibiotic resistance in V. cholerae has been reported to be due to mobile genetic elements and plasmid-mediated resistance.In our study setup, a V. cholerae Amp C producer strain due to the blaDHA gene and a carbapenemase producer due to the blaNDM-1 gene had been isolated in the past [35].

Trends in Aeromonas species
Aeromonas is an important but often neglected pathogen causing gastrointestinal diseases.It can mimic cholera.There have been several reports of increase in drug-resistant Aeromonas, especially with respect to beta-lactams, quinolones, and tetracyclines.In our study, maximum resistance was detected in Aeromonas species for ampicillin, though only a few species are known to be intrinsically resistant to it.Many of our isolates were found to be resistant to the third-generation cephalosporins, with the MIC for ceftriaxone being ≥4 µg ml −1 [36].The presence of blaCTX-M and inducible AmpC beta-lactamase (presence of MOX gene) have been documented in 70 and 24% of the isolates.Most commonly CTX M-15 is isolated in India.Fluoroquinolones have been the drug of choice but isolates that are resistant to nalidixic acid and susceptible to ciprofloxacin are known to possess the gyrA gene and hence can develop resistance to fluoroquinolones [37].
In our study, a cyclical trend was observed for fluoroquinolones.CphA harboured in common Aeromonas species such as A. hydrophila, A. dhakensis, A. jandaei, and A. veronii is the main chromosomal MBL recognized in aeromonads [38].Contaminated water sources can act as a medium for the transfer of antimicrobial-resistant genes.
In a study performed by Roman et al., higher susceptibility to tetracyclines was noted for Aeromonas species, similar to our study [39].However, in some Asian studies, tetracycline resistance was also noted [40][41][42].
In a study performed by Mohan et al. in northern India, resistance was observed to fluoroquinolones and co-trimoxazole for Aeromonas species, similar to our study [43].

Heterogeneity of the pathogens
Heterogeneity in terms of serotypes, resistotypes, or genotypes is an important factor observed in our diarrhoeal isolates of Shigella, Salmonella, and V. cholerae, as far as the survival of these pathogens is concerned.The extensive use of azithromycin led to the rise of drug-resistant strains of S. sonnei [44], a phenomenon observed in our study as well.Serotype switching among Shigella species is a well-known phenomenon and leads to the emergence of many untypable Shigella species, as documented in an earlier study [45].Our currently circulating strains of S. flexneri are of serotype 2a, followed by 6 and 3b; though some rare types such as type 1 variant and type 4 were also seen.O antigen loss (OmpA), an observation in a few of our S. flexneri strains [17], is another factor that can affect the conventional serotyping and may lead to the failure of an effective vaccine targeted against these antigens.Genetic heterogeneity was detected by CRISPR among our S. flexneri isolates; variations were substantial, and CRISPR type 3 was the most predominant [46].Chen et al., revealed that the presence of multiple insertion sequences in the cas genes lowered the activity of the CRISPR-Cas system, aiding the bacteria to acquire more extra genetic elements such as the AMR genes through horizontal gene transfer [47].
Salmonella, on the other hand, is known to show variations in its O and H antigens to escape immune clearance, often leading to either loss of these antigens or their masking, which can affect the conventional serotyping results.The emergence of thirdgeneration-resistant Salmonella isolates, both typhoidal and NTS, because of the rampant use of oral cefixime has probably led to this [48].
The major driving force in the heterogenous characteristics of these pathogens is horizontal gene transfer, depending on genomic plasticity, which leads to increasing AMR and selecting out strains/species in response.

Other bacterial pathogens isolated
Campylobacter species-level identification was performed in our setup using multiplex PCR targeting 16S rRNA (genus Campylobacter), mapA (C.jejuni), ceuA (C.coli), and actB for internal control [49].E. tarda infections may be attributed to the presence of a marine environment.P. shigelloides has been associated with ingestion of seafood, which is a common practice in coastal areas.

Bacterial and parasitic co-infections in our study
Some bacterial and parasitic co-infections were observed in our study.Shigella and Salmonella co-infection with the parasites was commonly noted.Parasites have certain mechanisms that they use to manipulate or evade the host immune response and cause infection [50].Parasites can alter the host's immune response and affect susceptibility to other infections.Normal gut flora affected by helminth can trigger the ability of bacteria to invade the intestine [51].

CONCLUSION
Diarrhoeal diseases pose a major threat in developing countries.They can be caused by a lack of proper sanitary facilities and access to safe drinking water.Limited access to laboratory facilities in developing countries forces clinicians to seek a syndromic approach and empirical use of broad-spectrum antibiotics, paving the way for the increasing number of drugresistant strains.Diarrhoeal pathogens such as Vibrio and Aeromonas are found freely in the environment and can survive in extreme conditions, hence the resistant traits can be passed on to various strains.Contaminated water can act as an agent for the transfer of antimicrobial resistance genes.Early diagnosis, targeted therapy, and proper antibiograms for diarrhoeal diseases can help in the reduction of resistant strains.Proper laboratory facilities for detecting the ESBL-producing strains and knowledge about the resistant genes prevailing in a geographical area can help to combat the spread of antimicrobial resistance among pathogens in the long run.

Table 1 .
Year-wise distribution of the common diarrhoeal pathogens

Table 2 .
Age-wise distribution of the common diarrhoeal pathogens

Table 5 .
Antibiotic susceptibility pattern of Vibrio cholerae* *Percentage susceptibility values are mentioned in the table.

Table 6 .
Antibiotic susceptibility pattern of Aeromonas species (n=80)* The number of susceptible isolates is mentioned in the table. *